This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Vitamin B6, the active cofactor of which is pyridoxal 52-phosphate (PLP), is an essential vitamin in the human diet. PLP is typically covalently bound to a PLP-dependent enzyme via an active site lysine until the substrate of that enzyme binds, when PLP is transferred to that ligand. PLP acts as an electron sink and helps to stabilize carbanion intermediates. This cofactor is known to be important in amino acid metabolism, and takes part in decarboxylation, transamination, and racemization reactions. Many structures are available of enzymes with PLP bound, and the structural biology of the biosynthesis of PLP has also been thoroughly studied. The degradation of PLP has been less well studied. Recently, the bacterium Mesorhizobium loti MAFF303099 has been shown to possess a pathway for producing succinic semialdehyde, ammonia, acetate, and carbon dioxide from PLP. The genes responsible for each of the seven enzymes of this catabolic pathway have been identified and the gene products kinetically characterized. The enzyme that catalyzes the sixth step, the oxidative ring opening of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) to produce E-2-(acetamidomethylene)succinate (E-2AMS), has been recently described. This enzyme is an FAD dependent enzyme, and the first of two structures has only FAD bound to 2.1 [unreadable]. The second complex contains both FAD and MHPC to 2.1 [unreadable]. This work has recently been published (McCulloch, 2009). The final enzyme of this pathway, E-2AMS hydrolase, is responsible for the hydrolysis of E-2AMS to produce ammonia, acetate, carbon dioxide, and succinic semialdehyde.